RNA viral diseases are responsible for the vast majority of viral morbidity and mortality of viral diseases of mankind, including AIDS, hepatitis, rhinovirus infections of the respiratory tract, flu, measles, polio and others. There are a number of chronic persistent diseases caused by RNA or DNA viruses that replicate through an RNA intermediate which are difficult to treat, such as hepatitis B and C, and T-cell human leukemia. Many common human diseases are caused by RNA viruses that are replicated by a viral encoded RNA replicase. Included in this group are influenza (Zurcher, et al., J. Gen. Virol. 77:1745 (1996), dengue fever (Becker, Virus-Genes 9:33 (1994), and rhinovirus infections (Horsnell, et al., J. Gen. Virol., 76:2549 (1995). Important RNA viral diseases of animals include feline leukemia and immunodeficiency, Visna maedi of sheep, bovine viral diarrhea, bovine mucosal disease, and bovine leukemia. Although some vaccines are available for DNA viruses, diseases such as hepatitis B are still prevalent. Hepatitis B is caused by a DNA virus that replicates its genome through a RNA intermediate (Summers and Mason, Cell 29:4003 (1982). While an effective vaccine exists as a preventive, there is no efficacious treatment for chronic persistent HBV infection.
Chain terminating nucleoside analogs have been used extensively for the treatment of infections by DNA viruses and retroviruses. These analogs are incorporated into DNA by DNA polymerases or reverse transcriptases. Once incorporated, they cannot be further extended and thus terminate DNA synthesis. Unfortunately, there is immediate selective pressure for the development of resistance against such chain terminating analogs that results in development of mutations in the viral polymerase that prevent incorporation of the nucleoside analog.
An alternative strategy is to utilize mutagenic deoxyribonucleosides (MDRN) or mutagenic ribonucleosides (MRN) that are preferentially incorporated into a viral genome. MDRN are incorporated into DNA by viral reverse transcriptase or by a DNA polymerase enzyme. MRN are incorporated into viral RNAs by viral RNA replicases. As a result, the mutations in the viral genome are perpetuated and accumulated with each viral replication cycle. With each cycle of viral infection, there ensues a chain like increase in the number of mutations in the viral genome. Eventually the number of mutations in each viral genome is so large that no active virally encoded proteins are produced.
5-aza-2′-deoxycytidine (5-aza-dC) is an antineoplastic agent that has been tested in patients with leukemia and is thought to act predominantly by demethylating DNA. 5-aza-cytidine (5-aza-C) has also been used to treat patients with leukemia. Methylation is thought to silence tumor growth suppressor and differentiation genes. Interestingly deamination of 5-aza-dC to 5-aza-2′-deoxyuridine (5-aza-dU) has been shown to result in loss of antineoplastic activity (see e.g., Momparler, et al., Leukemia. 11:1-6 (1997)).
5-aza-cytidine (5-aza-C) has also been used to treat patients with leukemia. Both 5-aza-C and 5-aza-dC were shown to inhibit HIV replication in vitro, although the mechanism of action was not determined (see e.g., Bouchard et al, Antimicrob. Agents Chemother. 34: 206-209 (2000)). More recently, 5-aza-C has been shown to be mutagenic to foot-and-mouth disease virus (see e.g., Sierra et al., J. Virol. 74(18): 8316-8323 (2000)). Both 5-aza-C and 5-aza-dC are unstable compounds. 5-aza-dC is rapidly degraded upon reconstitution. At pH 7.0, a 10% degradation occurs at temperatures of 25° C. and 50° C. after 5 and 0.5 hours, respectively (see e.g., Van Groeningen et al., Cancer Res. 46:4831-4836 (1986)). Thus, therapeutic use of 5-aza-C and 5-aza-dC is limited for treatment of both viral diseases and cancer. The present invention solves this and other problems.